The overwhelming success of blocking negative checkpoint regulators (NCR; e.g. CTLA-4, PD-1) in cancer has irrefutably validated NCRs as effective therapeutic targets in unleashing adaptive immunity in humans. We now present compelling evidence that V-domain Ig suppressor of T cells (VISTA), in addition to its role in regulating adaptive immunity, is a central negative regulator of innate immunity. Anti-VISTA mabs that trigger through VISTA disrupt neutrophil and macrophage chemotaxis, alter the myeloid transcriptome and program myeloid cells to an anti-inflammatory phenotype. Accordingly, anti-VISTA administration profoundly mitigates pathology in autoimmune disease models mediated by the innate immune system. This proposal will comprehensively address the impact of checkpoint regulation by VISTA on the genetics, epigenetics, biochemistry and immunology of myeloid-mediated inflammation. SA#1. VISTA is expressed on neutrophils at very high densities and we propose that VISTA is a central NCR of neutrophil activities. Data show that anti-VISTA triggering ablates their chemotactic activities to inflammatory chemokines and arrests neutrophil-mediated inflammation in vivo. A comprehensive analysis of VISTA negative regulation of neutrophil activation (chemotaxis, rolling, adhesion, NETs, etc.) will be performed. Unparalleled mechanistic insights into the therapeutic impact of anti-VISTA in the neutrophil-mediated K/BxN arthritis model will be provided by intravital microscopy in treated mice. SA#2. VISTA acts as an NCR in monocytes and the mechanisms by which VISTA re-programs their biology will be characterized. Within the macrophage lineage, we show that VISTA signaling directs macrophages to an anti- inflammatory program. Defined by either macrophage tolerance or enhanced M1M2 transition, VISTA elicits a well-defined transcriptional and functional program that is anti-inflammatory. We propose that triggering through VISTA can instruct myeloid reprogramming through the induction of a unique, VISTA-specific transcriptional profile that ablates myeloid chemotaxis, mediator production and inflammation. SA#3. Anti-VISTA ameliorates lupus by diminishing myeloid migration into the kidney and re-programming the inflammatory profile of infiltrating myeloid cells. A correlation between the therapeutic impact of anti-VISTA and the treatment-induced changes in myeloid heterogeneity and gene expression at the single cell level within the kidney will be established. Furthermore, a comprehensive understanding of how anti-VISTA alters chemokine-dependent positioning and the histological landscape of myeloid cells in the diseased kidney will be afforded by multiplex imaging of myeloid subsets in anti-VISTA treated NZB/NZW F1 mice. Together, these two approaches will offer unique insights into the negative regulation of innate immunity by VISTA in the pathogenesis of autoimmune kidney disease. In summary, the experiments outlined herein will solidify the emerging role of VISTA as a critical NCR in innate immunity.